The cultivation of agricultural crop plants serves mainly for the production of foodstuffs for humans and animals. Monocultures in particular, which are the rule nowadays, are highly susceptible to an epidemic-like spreading of diseases. The result is markedly reduced yields. To date, the pathogenic organisms have been controlled mainly by using pesticides. Nowadays, the possibility of directly modifying the genetic disposition of a plant or pathogen is also open to man. Alternatively, natural occurring fungicides produced by the plants after fungal infection can be synthesized and applied to the plants.
Resistance generally describes the ability of a plant to prevent, or at least curtail the infestation and colonization by a harmful pathogen. Different mechanisms can be discerned in the naturally occurring resistance, with which the plants fend off colonization by phytopathogenic organisms (Schopfer and Brennicke (1999) Pflanzenphysiologie, Springer Verlag, Berlin-Heidelberg, Germany).
With regard to the race specific resistance, also called host resistance, a differentiation is made between compatible and incompatible interactions. In the compatible interaction, an interaction occurs between a virulent pathogen and a susceptible plant. The pathogen survives, and may build up reproduction structures, while the host is seriously hampered in development or dies off. An incompatible interaction occurs on the other hand when the pathogen infects the plant but is inhibited in its growth before or after weak development of symptoms (mostly by the presence of R genes of the NBS-LRR family, see below). In the latter case, the plant is resistant to the respective pathogen (Schopfer and Brennicke, vide supra). However, this type of resistance is mostly specific for a certain strain or pathogen.
In both compatible and incompatible interactions a defensive and specific reaction of the host to the pathogen occurs. In nature, however, this resistance is often overcome because of the rapid evolutionary development of new virulent races of the pathogens (Neu et al. (2003) American Cytopathol. Society, MPMI 16 No. 7: 626-633).
Most pathogens are plant-species specific. This means that a pathogen can induce a disease in a certain plant species, but not in other plant species (Heath (2002) Can. J. Plant Pathol. 24: 259-264). The resistance against a pathogen in certain plant species is called non-host resistance. The non-host resistance offers strong, broad, and permanent protection from phytopathogens. Genes providing non-host resistance provide the opportunity of a strong, broad and permanent protection against certain diseases in non-host plants. In particular, such a resistance works for different strains of the pathogen.
Fungi are distributed worldwide. Approximately 100 000 different fungal species are known to date. Thereof rusts are of great importance. They can have a complicated development cycle with up to five different spore stages (spermatium, aecidiospore, uredospore, teleutospore and basidiospore).
During the infection of plants by pathogenic fungi, different phases are usually observed. The first phases of the interaction between phytopathogenic fungi and their potential host plants are decisive for the colonization of the plant by the fungus. During the first stage of the infection, the spores become attached to the surface of the plants, germinate, and the fungus penetrates the plant. Fungi may penetrate the plant via existing ports such as stomata, lenticels, hydatodes and wounds, or else they penetrate the plant epidermis directly as the result of the mechanical force and with the aid of cell-wall-digesting enzymes. Specific infection structures are developed for penetration of the plant. To counteract plants have developed physical barriers, such as wax layers, and chemical compounds having antifungal effects to inhibit spore germination, hyphal growth or penetration.
The soybean rust Phakopsora pachyrhizi directly penetrates the plant epidermis. After crossing the epidermal cell, the fungus reaches the intercellular space of the mesophyll, where the fungus starts to spread through the leaves. To acquire nutrients the fungus penetrates mesophyll cells and develops haustoria inside the mesophyl cell. During the penetration process the plasmamembrane of the penetrated mesophyll cell stays intact.
Fusarium species are important plant pathogens that attacks a wide range of plant species including many important crops such as maize and wheat. They cause seed rots, seedling blights as well as root rots, stalk rots and ear rots. Pathogens of the genus Fusarium infect the plants via infected seeds, roots or silks or they penetrate the plant via wounds or natural openings and cracks. After a very short establishment phase the Fusarium fungi start to secrete mycotoxins such as trichothecenes, zearalenone and fusaric acid into the infected host tissues leading to cell death and maceration of the infected tissue. Nourishing from dead tissue the fungus then starts to spread through the infected plant leading to severe yield losses and decreases in quality of the harvested grain.
Biotrophic phytopathogenic fungi depend for their nutrition on the metabolism of living cells of the plants. This type of fungi belong to the group of biotrophic fungi, like many rust fungi, powdery mildew fungi or oomycete pathogens like the genus Phytophthora or Peronospora. Necrotrophic phytopathogenic fungi depend for their nutrition on dead cells of the plants, e.g. species from the genus Fusarium, Rhizoctonia or Mycospaerella. Soybean rust has occupied an intermediate position, since it penetrates the epidermis directly, whereupon the penetrated cell becomes necrotic. After the penetration, the fungus changes over to an obligatory-biotrophic lifestyle. The subgroup of the biotrophic fungal pathogens which follows essentially such an infection strategy are heminecrotrohic.
Scopoletin and scopolin are antimicrobial phenolic hydroxycumarins that accumulate in different plants upon infection with various pathogens such as fungi or bacteria or in response to insect feeding damage, mechanical injury, dehydration or various other abiotic stresses.
Scopoletin shows broad antimicrobial activity and can inhibit development and growth of various fungi or bacteria in vitro (Goy, P. A., Signer, H., Reist, R., Aichholz, R., Blum, W., Schmidt, E., and Kessmann, H. (1993). Accumulation of scopoletin is associated with the high disease resistance of the hybrid Nicotiana glutinosa×Nicotiana debneyi. Planta 41: 200-206; Tal, B. and Robeson, D. J. (1986b). The Metabolism of Sunflower Phytoalexins Ayapin and Scopoletin: Plant-Fungus Interactions. Plant Physiology 82: 167-172.).
Scopoletin and its glucoside scopolin originate from the phenylpropanoid pathway (FIG. 1; (Kai, K., Mizutani, M., Kawamura, N., Yamamoto, R., Tamai, M., Yamaguchi, H., Sakata, K., and Shimizu, B. (2008). Scopoletin is biosynthesized via ortho-hydroxylation of feruloyl CoA by a 2-oxoglutarate-dependent dioxygenase in Arabidopsis thaliana. Plant Journal 55: 989-99).
Key steps of scopletin/scopolin synthesis comprise ortho hydroxylation of feruloyl-CoA, trans/cis isomeration of the side chain, lactonization and—considering scopolin synthesis—glycosylation (Kai et al., 2008). In Arabidopsis it has recently been shown that scopoletin production depends on ortho hydroxylation of feruloyl-CoA by the Fe(II)- and 2-oxoglutarate-dependent dioxygenase F6H1 (At3g13610). E-Z isomerisation of the side chain and lactonization were found to occur spontaneously. (Kai et al., 2008).
In planta accumulating scopoletin can finally be glucosylated to produce scopolin. Several Arabidopsis glucosyltransferases (e.g. UGT71C1) (Lim, E.-K., Baldauf, S., Li, Y., Elias, L., Worrall, D., Spencer, S. P., Jackson, R. G., Taguchi, G., Ross, J., and Bowles, D. J. (2003). Evolution of substrate recognition across a multigene family of glycosyltransferases in Arabidopsis. Glycobiology 13: 139-45.) as well as two different tobacco glucosyltransferases (Togt1 and Togt2) (Fraissinet-Tachet, L., Baltz, R., Chong, J., Kauffmann, S., Fritig, B., and Saindrenan, P. (1998). Two tobacco genes induced by infection, elicitor and salicylic acid encode glucosyltransferases acting on phenylpropanoids and benzoic acid derivatives, including salicylic acid. FEBS letters 437: 319-23) have been identified that can catalyze glycosylation of scopoletin in vitro.
Scopolin is generally regarded a less potent antimicrobial agent than scopoletin. Following pathogen-induced mechanical injury or hypersensitive reactions (HR), decompartimentalization of scopolin containing cells might lead to the release of scopolin from vacuoles into the cytoplasm and subsequent hydrolysis of the glucose conjugate by β-glucosidases.
Scopoletin and its glucoside scopolin are widely distributed among the plant kingdom and have been detected in various plant organs of approximately 80 different plant families. Interestingly, scopoletin biosynthesis seems to be lost in several economically important crops (e.g. Glycine max, Zea mays, Triticum aestivum, Oryza sativa etc.), indicating that the ability to synthesize this antimicrobial substance might have been lost during breeding. However, this does not apply to sweet potato, tobacco, sunflower, cotton or cassava since scopoletin has been shown to accumulate in these crops in response to infection (summarized by Gnonlonfin, G. J. B., Sanni, A., and Brimer, L. (2012). Review Scopoletin—A Coumarin Phytoalexin with Medicinal Properties. Critical Reviews in Plant Sciences 31: 47-56).
Soybean rust has become increasingly important in recent times. The disease may be caused by the biotrophic rusts Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur). They belong to the class Basidiomycota, order Uredinales, family Phakopsoraceae. Both rusts infect a wide spectrum of leguminosic host plants. P. pachyrhizi, also referred to as Asian rust, is the more aggressive pathogen on soy (Glycine max), and is therefore, at least currently, of great importance for agriculture. P. pachyrhizi can be found in nearly all tropical and subtropical soy growing regions of the world. P. pachyrhizi is capable of infecting 31 species from 17 families of the Leguminosae under natural conditions and is capable of growing on further 60 species under controlled conditions (Sinclair et al. (eds.), Proceedings of the rust workshop (1995), National SoyaResearch Laboratory, Publication No. 1 (1996); Rytter J. L. et al., Plant Dis. 87, 818 (1984)). P. meibomiae has been found in the Caribbean Basin and in Puerto Rico, and has not caused substantial damage as yet.
P. pachyrhizi can currently be controlled in the field only by means of fungicides. Soy plants with resistance to the entire spectrum of the isolates are not available. When searching for resistant soybean accessions, six dominant R-genes of the NBS-LRR family, which mediate resistance of soy to P. pachyrhizi, were discovered. The resistance was lost rapidly, as P. pachyrhizi develops new virulent races.
Increasing resistance to Fusarium is one of the most important goals in maize breeding. Despite having a great natural diversity in interaction phenotypes with Fusarium species, resistance seems to be distributed over many weak QTLs with low heritability. Therefore only little progress was made in increasing resistance against Fusarium by breeding.
In recent years, fungal diseases, e.g. soybean rust and Fusarium graminearum have gained in importance as pest in agricultural production. There was therefore a demand in the prior art for developing methods to control fungi and to provide fungal resistant plants.
Much research has been performed on the field of powdery and downy mildew infecting the epidermal layer of plants. However, the problem to cope with soybean rust which infects the mesophyll or Fusarium fungi that infect inaccessible inner tissues remains unsolved.
The object of the present invention is inter alia to provide a method of increasing resistance against fungal pathogens, preferably against fungal pathogens of the family Phakopsoraceae, more preferably against fungal pathogens of the genus Phakopsora, most preferably against Phakopsora pachyrhizi (Sydow) and/or Phakopsora meibomiae (Arthur), also known as soybean rust.
A further object of the present invention is inter alia to provide a method of increasing resistance against fungal pathogens, preferably against fungal pathogens of the genus Fusarium, most preferably against Fusarium graminearum and/or Fusarium verticillioides. 
Surprisingly, we found that fungal pathogens, in particular of the genus Phakopsora, for example soybean rust and/or of the genus Fusarium, for example Fusarium graminearum and/or Fusarium verticillioides, can be controlled by increased production or increased accumulation of scopoletin or derivatives thereof in a plant and by direct application of scopoletin or derivatives thereof to the plant.
Surprisingly, we found that fungal pathogens, in particular of the genus Phakopsora, for example soybean rust and of the genus Fusarium, for example Fusarium graminearum and/or Fusarium verticillioides, can be controlled by increased expression of the F6H1 protein, optionally in combination with one or more proteins selected from the group consisting of CCoAOMT1, ABCG37 and UGT71C1.
The present invention therefore provides a method of increasing resistance against fungal pathogens, preferably against fungal pathogens of the family Phakopsoraceae and/or Nectriaceae, more preferably against fungal pathogens of the genus Phakopsora and/or Fusarium, most preferably against Phakopsora pachyrhizi (Sydow), Phakopsora meibomiae (Arthur), Fusarium graminearum and/or Fusarium verticillioides in transgenic plants, plant parts, or transgenic plant cells by increasing the production and/or accumulation of scopoletin and/or derivatives thereof or by exogenous application of scopoletin and/or derivatives thereof to plants, plant parts, or plant cells.
A further object is to provide transgenic plants resistant against fungal pathogens, preferably of the family Phakopsoraceae and/or Nectriaceae, more preferably against fungal pathogens of the genus Phakopsora and/or Fusarium, most preferably against Phakopsora pachyrhizi (Sydow), Phakopsora meibomiae (Arthur), Fusarium graminearum and/or Fusarium verticillioides, a method for producing such plants as well as a recombinant vector construct useful for the above methods.
The present invention also refers to a recombinant vector construct and a transgenic plant, plant part, or plant cell comprising exogenous nucleic acids or fragment thereof which lead to enhanced production of scopoletin and/or derivatives thereof. Furthermore, a method for the production of a transgenic plant, plant part or plant cell using the nucleic acids of the present invention is claimed herein. In addition, the use of a nucleic acid or the recombinant vector of the present invention for the transformation of a plant, plant part, or plant cell is claimed herein.
The present invention also refers to method for applying a scopoletin and/or derivatives to a surface of a plant, plant part or plant cell as well as plant surface or plant part surface coated with scopoletin and/or derivatives.
The objects of the present invention, as outlined above, are achieved by the subject-matter of the main claims. Preferred embodiments of the invention are defined by the subject matter of the dependent claims.